The MS Facility has been active in developing methodology using MALDI-MS to characterize phosphorylated proteins. The information desired includes the number of phosphorylation sites and their locations. The analysis is performed by digesting a phosphorylated peptide, using MALDI-MS to analyze the digestion, comparing the results with computed possibilities using software developed in-house, and subjecting the digestion mixture to further degradation using alkaline phosphatase to confirm assignments of peaks representing phosphorylated protein fragments. While the process as developed has been successfully used, aspects remain to be optimized. This work is being pursued using _-Casein as a model phosphoprotein. Two aspects are focal points. The first is related to the relatively low responses observed in MALDI-MS for phosphopeptides relative to unmodified peptides. This is an important problem, since the information desired is usually found in the smaller peaks in the MALDI mass spectra. Relative responses of phosphorylated and unphosphorylated peptides are being characterized for a variety of matrices, with specific interest in the negative ion mode of MALDI-MS, to improve analytical capabilities. The second aspect is related to the purity and singularity of phosphopeptides such as _-Casein. Are pure samples usually encountered, or is there usually substantial variability? High resolution two dimensional gel electrophoresis is being used to determine the number of phosphorylation variants of the peptide that exist, as well as providing a means for separating them and characterizing them separately.